Questions & Answers

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General

Which species are compatible?

The Ribo-Zero kits were developed using model organisms. Results can vary depending on the species from which the RNA sample was derived, especially with 5S rRNA removal.

Visit the RNAMatchMaker tool, www.epibio.com/rnamatchmaker, on the Epicentre website to confirm species compatibly.

How do I know if my species works with a particular Ribo-Zero kit?

See our species compatibility tables. If you do not see your species in the list please use RNAMatchMaker to learn if a current Ribo-Zero kit will match your organism.

Can I get the probe sequences for Ribo-Zero?

No. Our sequences are proprietary and we are unable to share them.

What is the difference between the standard Ribo-Zero kit and the Ribo-Zero Gold kit?

The Ribo-Zero Gold Kit contains additional capture probes that cover mitochondrial rRNA sequences, which account for about 7% of the rRNA in most mammalian cells. The standard Ribo-Zero Kit will remove cytoplasmic (nuclear-encoded) rRNA, but not mitochondrial rRNA.

Do Ribo-Zero kits only remove rRNA?

Yes. While there is a possibility of some cross-hybridization between the capture probes and minute portions of other RNA species, the hybridization conditions are optimized for rRNA capture while minimizing undesirable cross-hybridization.

Why is the Ribo-Zero rRNA depletion method better for mRNA enrichment compared to poly(A) enrichment techniques, especially for degraded/FFPE samples?

Poly(A) enrichment involves the capture of the 3´-end of polyadenylated RNA, typically using oligo(dT) columns. If the RNA to be enriched is of high quality (RIN >9), then most of the mRNA will be recovered. However, if the input RNA is of poor quality (RIN <6), then the majority of the mRNA sequences will be lost and the resulting mRNA-Seq libraries will contain only 3´ sequences of the mRNA. By using the Ribo-Zero procedure, capture of the rRNA (intact or degraded) does not affect the mRNA sequences in the original RNA preparation. Libraries prepared after Ribo-Zero treatment therefore exhibit more even coverage density, and greatly reduced 3´ end bias compared to poly(A) enrichment. R-Z treatment also recovers non-poly(A), non-rRNA such as lincRNA that Poly(A) enrichment loses.

Can any of the Ribo-Zero kits be used with environmental microbial samples?

Yes. The Ribo-Zero Bacteria Kit contains capture probes for both Gram-positive and Gram-negative bacteria, thus allowing removal of rRNA from an RNA sample that contains transcripts from many bacterial species.

Do you still sell the older, nonmagnetic Ribo-Zero kits?

No. We retained the non-magnetic (filter-based) “Core Kits” until December 2015 and are currently no longer available

Input

How much input RNA is too much?

With the exception of the Epidemiology kit, Ribo-Zero kits support 1–5 µg total RNA for standard input or 100 ng to 1 µg total RNA for low input. Do not exceed the maximum amount of RNA. Quantify the input total RNA using a Qubit Fluorometer. The RNA concentration reported by the Bioanalyzer or NanoDrop might not be accurate.

Can the Ribo-Zero kit deplete degraded samples?

Ribo-Zero kits remove rRNA from partially degraded samples. However, probes cannot hybridize to severely degraded samples and are therefore not effective.

How do I manage DNA contamination?

RNA samples must be trated with DNase before starting the rRNA removal protocol. Otherwise, the probes can bind to the DNA and reduce rRNA removal.

I don’t have 1 μg of Total RNA. Do you have kits for a low input amount?

You can use as low as 100 ng in the standard Ribo-Zero Kits. Just follow the instructions for 1-2.5 µg input listed in the User's Guide.

How do I remove rRNA from a mix of mammals, plants, and bacteria, etc.?

You can purchase a kit for each of the organisms and then mix the rRNA removal solution in an equal ratio. Then proceed with the protocol as stated in the user guide.  You may want to decrease your input amount to 2.5 ug to ensure the rRNA is removed fully.

How well do the Ribo-Zero Kits perform with FFPE samples?

The Ribo-Zero Kits work very well on FFPE RNA. The design and configuration of the probes allows for the capture of very small pieces of rRNA, leaving the mRNA and other non-ribosomal species in the supernatant.

Protocol

How does the Ribo-Zero procedure work?

Ribo-Zero kits remove ribosomal RNA (rRNA) using a hybridization/bead capture procedure that selectively binds rRNA species using biotinylated capture probes. The probe:rRNA hybrid is then captured by magnetic beads and removed using a magnet, leaving the desired rRNA-depleted RNA in solution.

How much mixing is required?

Follow the protocol as described in the reference guide for your kit. The procedures require frequenent and complete mixing for successful rRNA removal.

How much removal solution do I use?

The appropriate volume of removal solution depends on the amount of total RNA input. Accurately quantify the amount of RNA and see the kit refence guide for instructions on determining the correct volume of solution.

At what temperature do I use the magnetic beads?

The magnetic beads must be at room temperature before washing. After washing, the beads must be kept at room temperature for use in rRNA removal.

How do I prevent the magnetic beads from freezing in storage?

If frozen, the magnetic beads do not work. Confirm that the beads were not frozen when they arrived in the lab, and that they were properly stored at 2°C to 8°C.

How do I mix the RNA sample with the magnetic beads?

Make sure that you add the RNA sample to the washed, room-temperature magnetic beads.  Do not add magnetic beads to the RNA sample.

What can I do about clumped magnetic beads?

Magnetic beads can clump after the probe-hybridized RNA sample is added. This happens because the probes contain biotin that can bind to multiple magnetic bead particles. Make sure that you vortext the beads vigorously for at least 10 seconds.

How do I make sure that the magnetic beads are completely removed?

Failure to remove the magnetic beads completely causes carryover of the  probes and rRNA. Place the tubes on a magnetic stand for 5 minutes, and then carefully transfer the supernatant containing the rRNA-depleted RNA to a new RNase-free tube.

If I accidentally freeze my magnetic beads, can I still use them?

No, the beads will no longer work.  You must purchase a replacement kit.

My Total RNA concentration is too low. Can I add more sample volume to the reaction?

No, please speed vac your sample to the appropriate volume but do not dry the sample down completely.

Do I need to have clean/purified RNA before I use Ribo-Zero or will it work in the buffer from my previous reaction?

The RNA needs to be purified for Ribo-Zero to work properly. If you use RNA that is not clean the salts will change the conditions of the reaction in the Ribo-Zero kit.

Library Evaluation

What do Bioanalyzer traces look like before and after Ribo-Zero treatment?

Ribo-Zero Bioanalyzer

Figure 1.  A. S. cereviseae samples from 2 mm colonies on solid YPD media was purified using the MasterPure™ Yeast RNA Purification Kit with DNase treatment, to achieve final yield of 3 µg RNA. B. 1 µg of purified RNA was processed using the Ribo-Zero™ Magnetic Gold Kit (Yeast) to remove the 18S and 25S rRNA. Results are similar with other organisms

Why did I noticed a peak at 100-140 nt in my Bioanalyzer traces after using a Ribo-Zero kit?

This is normal and expected. The peaks observed in the 100- to 140-nt region on a Bioanalyzer trace correspond to tRNA, small noncoding RNAs, and small amounts of 5S rRNA (in cases where 5S rRNA removal is less efficient). These small RNAs are available for inclusion in a sequencing library if desired, but can be readily removed by gel or other size-selection techniques that are designed to remove small RNAs, either before or after library synthesis.